2025-08-28
ENA first public
ERC000011
ENA-CHECKLIST
ITB-CNR, Institute of Biomedical Technologies, National Research Council, Segrate, Italy  INGM Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy
INSDC center name
public
INSDC status
ERS25489018
SRA accession
E-MTAB-14226_3:SAMMY_CTR1_S2L
Submitter Id
age at diagnosis
derived time unit
age
62
year
age
ArrayExpress
broker name
not collected
collection date
human
common name
Protocols: Our cohort includes chemo-naïve patients followed in the Urology Division of Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico (Milan) who underwent the transrectal ultrasound-guided systematic sampling of prostate tissue (TRUS biopsy). This diagnostic procedure was performed as part of routine clinical management following the detection of abnormal digital rectal examination or an elevated PSA blood level. According to the current standard for the detection of PCa, 14 ultrasound-guided biopsy cores (diagnostic biopsies), seven from each side of the prostate gland, were collected from each patient for the clinical diagnosis. During the same procedure, one additional biopsy core to be used for our research project (research biopsy), was taken from a site directly adjacent to one of the diagnostic biopsies. Samples selected for epigenome studies consist of 17 fresh biopsies divided on the bases of the histology and the spatial distribution of the positive cores in two different groups: 10 PCa biopsies with histologically confirmed prostate cancer and 7 who had no cancer in any biopsy core.  Chromatin fractions (S2, S3 and S4) were diluted 1:2 in 1X TE buffer (10mM TrisHCl pH 8.0, 1 mM EDTA) and incubated with 3 U of RNAse cocktail (Ambion, AM2286) at 37° for 90 minutes, followed by 40μg of Proteinase K (Invitrogen, AM2548) at 55° for 150 minutes. Genomic DNA was then isolated using phenol/chloroform (Sigma-Aldrich, 77617) extraction, followed by a back extraction of phenol/chloroform with additional volume of TE1X. DNA was precipitated in 2 volumes of cold ethanol, 0.3M sodium acetate and 20ug glycogen (Ambion AM9510) for 1 hour on dry ice or overnight at -20°C. Dry pellets were resuspended in 50 ul (S2) or 15 ul (S3 and S4) of nuclease-free water and incubated at 4°C overnight. On the next day, S2 was further purified using PCR DNA Purification Kit (Qiagen, 28106) and separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) with the ratio 0.90/0.95 to obtain smaller fragments conserved as S2S (< 300 bp) and larger fragments labelled as S2L (> 300bp) fractions. Both were resuspended in 20 ul of nuclease-free water and then reduced to 15ul using a centrifugal vacuum concentrator. S2L, S3 and S4 fractions were sonicated in a Covaris M220 focused-ultrasonicator using screw cap microTUBEs (Covaris, 004078) to obtain a smear of DNA fragments peaking at 150-200 bp (waterbath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50, 150 seconds for S2L and 175 seconds for S3 and S4). Fractions were quantified using Qubit 4 fluorometer with Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). Libraries were generated from each sample using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and Unique Dual Index NEBNextMultiplex Oligos for Illumina (NEB, E6440S). Libraries were then qualitatively and quantitatively checked on and run on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626).
description
adult
developmental stage
Healthy Biopsy
disease
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
prostate gland
organism part
P28
original sample name
Homo sapiens
scientific_name
male
sex
SAMMY_CTR1_S2L
title